Numerous medical conditions require treatment via injection of gamma globulin. The manner of preparation of the gamma globulin is of critical importance, particularly in order to eliminate the chance of contracting viral hepatitis. Viral hepatitis is a debilitating disease at best and lethal at worst. Consequently, any advances made to eliminate the chance of contamination of any injectable product by this virus are of immense importance.
Hepatitis B virus is estimated to infect approximately 200 million persons worldwide. Since the base material for the production of gamma globulins often is plasma obtained from a human source, the chances of obtaining contaminated or infected plasma are significant in view of the substantial number of persons who are carriers, chronically infected and acutely infected. The infected plasma from such a person may contain not only varying amounts of viral particles but also different sizes and forms of the particles. One common form is the spherical particle which has a mean diameter of 22 nm. These spherical particles are generally devoid of DNA and represent free envelopes of the virus. Less common are the 42 nm Dane particles which represent the virion and consist of an envelope and a 27 nm nucleocapsid that contains a molecule of DNA. Free nucleocapsids may be observed in the nucleus of infected hepatocytes but are generally not found in the plasma. Infected hepatocytes have been found to synthesize excessive amounts of envelope having a half life of 3.3 days and can be found circulating throughout the body. In accordance with the different components of viral particles, different immunological markers have been identified. For example, associated with the core is an antigen commonly labelled HB.sub.c Ag and an "e" antigen labelled HB.sub.e Ag. The most common antigen employed for the detection of hepatitis B virus infection, however, is the surface antigen HB.sub.s Ag.
HB.sub.s Ag surface antigens are generally thought to be associated with the envelopes. Since immunoglobulins selective for the heptitis B surface antigen are protective against hepatitis B infection, as a consequence, the virus free envelopes present in the plasma of chronic carriers can effectively be used as a source of a vaccine. The structure and genetic organization of the hepatitis viral particle has been reviewed in an article by Tiollais et al., in Biology of Hepatitis B Virus, Science, Vol. 213, 406-411 (July, 1981). Further discussion concerning the association of the Australian antigen with persistent or chronic hepatitis may be obtained in Australia Antigen and Hepatitis by Blumberg et al., C.R.C. Monotopic Series, C.R.C. Press, Cleveland, Ohio (1982). The close relationship of hepatitis B surface antigen to hepatitis viral infectivity is discussed by Blumburg, supra, on page 14.
Historically, a number of tests have been developed for the testing and identification of hepatitis-type viral infections and are uniformly directed towards the detection of hepatitis related antigens or the antibodies specific therefor. These tests have generally been characterized as a first, second or third generation test depending upon their sensitivity in the detection of weakly positive HB.sub.s Ag reference panel samples obtainable from the U.S. Bureau of Biologics. Presently, the most sensitive tests available are of the third generation category and include radioimmunoassay, enzyme linked immunosorbent asssay, reversed passive hemagglutination and reversed passive latex agglutination tests. These third generation hepatitis tests are typically capable of detecting 10.sup.9 HB.sub.s Ag particles per ml of serum. Unfortunately, an ml of serum need contain only approximately 10.sup.6 -10.sup.7 HB.sub.s Ag particles per ml in order to be infective. It thus becomes readily apparent that even a negative result with the most sensitive test available will fail to ensure the noninfectivity of a sample. Consequently, the manner of preparation for an immunoglobulin injectable reagent from a potentially infected plasma source becomes of paramount importance since any production method should ideally be capable of removing substantially all infective viral particles. At present, only in vivo chimpanzee studies are sufficiently sensitive to ensure noninfectivity of any particular sample. The cost and the requirements of such studies make them prohibitive for routine use. See "The Test for HB-Associated Antigens and Antibodies" by Gerety et al. in Chapter 11 of Viral Hepatitis, Ed. Vyas et al., The Franklin Institute Press, Philadelphia, PA (1978).
Presently, all immunoglobulin injectable materials approved for use by the FDA and Bureau of Biologics have been produced by the alcohol fractionation procedure developed by Dr. E. Cohn of Harvard during the 1940s and described in Cohn et al., J. Am. Chem. Sos., 68, 459 (1946). This procedure, coupled with the careful selection of plasma negative for hepatitis infectivity, determined by the most sensitive tests available, has been employed for such a long period of time that the U.S. government has adopted a position favoring only the resultant preparations of this procedure as safe. That the products produced by this procedure are indeed safe can easily be demonstrated by the millions of non-infected recipients of product. Unfortunately, occasional problems still arise demonstrating that despite the favorable appearance of the `numbers` correlated with the Cohn process, the Cohn process still will not ensure complete noninfectivity. Many investigators have pointed to the development of both a plasma screening process and increasingly sensitive detection tests (to thereby eliminate source plasma having questionable infectivity) as the reason by the apparent success of the Cohn process. Despite the apparent success of the Cohn process, there is great economic pressure to develop superior production methods. The Cohn process is disadvantageous because vast volumes of plasma are required. Plasma is not only expensive but is also present only in limited supply.
It is an object of the present invention to provide a process whereby hepatitis B surface antigens may be safely eliminated from a plasma by a more efficient process than that developed by Cohn et al. The removal of HB.sub.s Ag is used as a measure since it is the most highly correlated indicator of hepatitis infectivity.
Hepatitis viral particles, useful for the preparation of a vaccine, cannot be adequately grown in tissue culture and must therefore be isolated from the blood of infected persons. Purification of the hepatitis antigen from the blood is necessary in order to remove contaminating blood components which would otherwise give rise to various serum sicknesses.
Such a method for the purification of hepatitis B surface antigen is described by Mizuno et al. in U.S. Pat. No. 4,162,192. Mizuno's process involves the passage of conventionally obtained sera, hopefully containing the hepatitis B surface antigen through an anion exchange chromotography column. The column adsorbs most of the blood plasma components but allows the HB.sub.s Ags to pass freely. The effluent from this column is then subsequently passed through a cation exchanger whereby the gamma globulins are adsorbed thus leaving only the desired hepatitis B surface antigens contained in the effluent. Passage of the infected sera through the anion and cation columns provides a solution containing hepatitis B surface antigens with most blood components removed.
It is another object of the present invention, rather than provide a solution containing HB.sub.s Ag free of gamma globulins, provide, instead a solution containing gamma globulins wherein all of the hepatitis B surface antigens are removed to thereby provide a safe, injectable immunoglobulin reagent.
Several conventional methods for the separation of gamma globulin from human serum have been described notably for Baumstark et al. in "A Preparative Method For The Separation of 7S Gamma Globulin From Human Serum", Archives of Biochemistry and Biophysics, 108, 514-522 (1964) and by A. J. Webb in "A 30-Minute Preparative Method For Isolation Of IgG From Human Serum", Vox Sang, 23:279-290 (1972). Although both of these papers are more concerned with the separation and selection of various gamma globulin classes from a serum containing numerous other contaminating proteins, they do address the removal of contaminating proteins and materials from the original serum sample. Both employ a DEAE-Sephadex column chromatographic material with a phosphate buffer eluting agent. Both investigators met with some degree of success as far as removal of contaminating proteins was concerned, however, both failed to address the problem of removing contaminating hepatitis viral particles in order to provide a safe, injectable reagent.
It is yet another object of the present invention to provide methods utilizing chromatographic column resin/buffer combinations which are more effective in removing contaminating hepatitis viral particles than those provided by conventional methods.
Another method, described by Stanworth in an article entitled "A Rapid Method Of Preparing Pure Serum Gamma Globulin", Nature, 188, 156-157 (1960), involves the use of a diethyl amino ethyl cellulose anion exchanger to remove proteins from human serum dialyzed to remove high molecular weight proteins, however, the method described fails to account for the effect on hepatitis viral contaminants and additionally fails to provide an injectable reagent, both of which are objects of the present invention.
Condie has described in U.S. Pat. No. 4,136,094, "Preparation of Intravenous Human and Animal Gamma Globulins And Isolation Of Albumin", another method for obtaining gamma globulin which is claimed safe for intravenous administration. Condie's method involves three manipulations including plasma stabilization by treatment with fumed colloidal silica, isolation and elution of gamma globuin and albumin from ion exchange resins and finally concentration dialysis and sterile filtration. The fumed colloidal silica step is provided to remove hepatitis associated antigen present in the plasma as well as a number of proteolytic enzymes and their precursors. The colloidal silica treated materials were tested for presence of hepatitis associated antigen by radioimmunoassay. The materials tested negative and intravenous administration of large quantities (in excess of 30 g) in over 50 patients showed no evidence of passage of hepatitis virus nor produced cases of hepatitis. To be noted, however, as previously discussed, testing by presently available radioimmunoassay procedures will not ensure that the tested sample is free of infective hepatitis. Without further testing, any such material will not be approved by the U.S. government for widespread use in excess of that required for limited clinical studies.
It is still another object of the present invention to provide a significantly simpler, effective procedure for isolating immunoglobulins from blood serum and hepatitis associated antigens which does not require fumed colloidal silica.
Treatment of hemolytic disease of the fetus or newborn has become rather standard and is accomplished by treatment of the mother by injection of Rho (D) immunoglobulin of human origin. Such a product is RhoGAM, available from the assignee hereof, operates by preventing the unimmunized Rho (D) negative mother from responding to Rho (D) antigen present on red cells and `received` at delivery from an Rho (D) positive infant. Thus, by preventing anti-Rho (anti-D) production by the mother at delivery, the subsequent Rho (D) positive infant of this mother is protected from hemolytic disease of the newborn. Although this successful product is presently produced by a Cohn alcohol fractionation type process, several investigators have attempted to use alternative methods to produce similar materials to thereby provide an economically more advantageous product, to reduce large plasma requirements and to obtain superior protection against the threat of hepatitis infection. Such investigational efforts have been reported by Hoppe et al. in "Prevention of Rh Immunization Modified Production of IgG Anti-Rh For Intravenous Application By Ion Exchange Chromatography", Vox Sang, 25:308-316 (1973) and Friesen et al. in "Column Ion-Exchange Preparation and Characterization of an Rh Immune Globulin for Intravenous Use", Journal of Applied Biochemistry, 3, 164-175 (1981).
Hoppe in Germany and Friesen in Canada both employed a DEAE-Sephadex chromatography column in conjunction with a phosphate buffer eluding agent. Hoppe's source of anti-D containing plasma was from volunteers who passed an HB.sub.s Ag laboratory test for at least six months, the plasma being stored in the interim. Thus, Hoppe employed a relatively safe, noninfective plasma to start with. No additional tests were run, however, to determine the efficacy of the DEAE-Sephadex resin/phosphate buffer combination for the removal of hepatitis B surface antigen. Hoppe's concern was instead directed towards the removal of aggregated materials and the isolation of an unfragmented, immunoelectrophoretically pure IgG having a relatively high antibody concentration. The Freisen publication reports on the modifications made to the Hoppe method for the development of an intravenous Rh IgG for use in Canada. As Hoppe had done, Freisen tested each unit of Rh plasma for HB.sub.s AG to eliminate any donors testing positive. Freisen employed the radioimmunoassay kit from Abbott Laboratories, North Chicago, Ill. (Ausria II Kit). This test is still regarded as one of the most sensitive and was also employed in the development of the invention described later. Freisen reported that clinical trials showed the material produced using the DEAE-Sephadex resin/phosphate buffer combination was effective and safe for the prevention of Rh immunization. Freisen, however, reported no additional tests for determining the efficacy of the DEAE-Sephadex/phosphate buffer combination for removing hepatitis B surface antigen from plasma samples. This, at least from the U.S. government's perspective, is especially important since the radioimmunoassay test employed in screening the donor plasma samples is incapable of detecting concentrations of HB.sub.s AG particles two or three orders of magnitude lower which may still be infective. It is this concern for the potential infectivity of a reagent produced by such a method that the United States government has been significantly more restrictive in permitting the production of injectable immunoglobulin reagents by solid phase methodologies.
It is an object of the present invention to provide resin/buffer systems that are superior in their ability to eliminate hepatitis B surface antigen than those employed by Hoppe or Friesen.